The epithelial Na channel (ENaC) provides for sodium entry across the apical membranes of many absorptive epithelia. In the kidneys, lungs, intestines and other tissues, these channels constitute a Na-selective entry path, whose importance in fluid and electrolyte homeostasis is highlighted by genetic diseases in which mutations of ENaC disturb fluid balance. This channel was recently cloned, so that its structural properties can now be related to its function in Na entry. Key features in ENaC regulation, which for decades have been the subject of numerous investigations, will emerge from knowledge of the channel's structure and its interactions with other proteins. We have recently identified a functional and physical interaction between ENaC and the membrane traffic regulatory protein, syntaxin. Our results identify a novel regulatory mechanism for the control of Na entry which may be analogous to the regulation of voltage- gated Ca channels at nerve terminals. We propose to pursue these findings by determining the mechanism of syntaxin interaction with ENaC, and its specificity for various syntaxin isoforms. We will determine whether syntaxin alters the insertion or retrieval processes that determine the number of functional Na Channels in the plasma membrane. We will identify the site of physical interaction between syntaxin and the three subunits of which functional Na channels are comprised and perform structure-function studies to resolve the sites in both ENaC and syntaxin which mediate these interactions. The ability of syntaxin to regulate the rate of Na entry across polarized epithelia will be determined by identifying the syntaxin isoforms expressed in A6 epithelia and assessing the effect of manipulating syntaxin expression on Na absorption. Proteins which regulate the activity of syntaxin, such as SNAP-25 and munc-18, and a protein implicated in ENaC degradation, Nedd4, will be examined for their influence on syntaxin-ENaC interactions. These studies will define the importance of a new regulatory mechanism for controlling Na entry in salt-absorbing epithelia. This process has important implications for the regulation of body salt and water homeostasis, the control of blood pressure and cardiovascular diseases.